Methods for reprogramming somatic cells

ABSTRACT

The invention provides methods for reprogramming somatic cells to generate multipotent or pluripotent cells. Such methods are useful for a variety of purposes, including treating or preventing a medical condition in an individual. The invention further provides methods for identifying an agent that reprograms somatic cells to a less differentiated state.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of U.S. ProvisionalApplication No. 60/525,612, filed Nov. 26, 2003, and U.S. ProvisionalApplication No. 60/530,042, filed Dec. 15, 2003, the specifications ofboth of which are incorporated herein by reference in its entirety.

GOVERNMENTAL FUNDING

The invention described herein was supported, in whole or in part, byGrant R37 CA84198 from the National Institutes of Health. The UnitedStates government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Pluripotent stem cells have the potential to differentiate into the fullrange of daughter cells having distinctly different morphological,cytological or functional phenotypes unique to a specific tissue. Bycontrast, descendants of pluripotent cells are restricted progressivelyin their differentiation potential, with some cells having only onefate. Pluripotent cells have extraordinary scientific and therapeuticpotential, as they can be differentiated along the desireddifferentiation pathway in a precisely controlled manner and used incell-based therapy.

Two categories of pluripotent stem cells are known to date: embryonicstem cells and embryonic germ cells. Embryonic stem cells arepluripotent stem cells that are derived directly from an embryo.Embryonic germ cells are pluripotent stem cells that are deriveddirectly from the fetal tissue of aborted fetuses. For purposes ofsimplicity, embryonic stem cells and embryonic germ cells will becollectively referred to as “ES” cells herein.

ES cells are presently obtained via several methods. In a first method,an ES cell line is derived from the inner cell mass of a normal embryosin the blastocyst stage (See U.S. Pat. No. 6,200,806, Thompson, J. A. etal. Science, 282:1145-7, 1998 and Hogan et al., 2003). A second methodfor creating pluripotent ES cells utilizes the technique of somatic cellnuclear transfer (SCNT). In this technique, the nucleus is removed froma normal egg, thus removing the genetic material. Next, a donor diploidsomatic cell is placed next to the enucleated egg and the two cells arefused, or the nucleus is introduced directly into the oocyte bymicromanipulation. The fused cell has the potential to develop into aviable embryo, which may then be sacrificed to remove that portion ofthe embryo containing the stem cell producing inner cell mass.

In a third method, the nucleus of a human cell is transplanted into anentirely enucleated animal oocyte of a species different from the donorcell (referred to herein as animal stem cell nuclear transfer, or“ASCNT”). See U.S. Pat. application Ser. No. 20010012513 (2001). Theresultant chimeric cells are used for the production of pluripotent EScells, in particular human-like pluripotent ES cells. One disadvantageof this technique is that these chimeric cells may contain unknownnon-human viruses and still contain the mitochondria of the animalspecies. Thus, there would be substantial risks of immune rejection ifsuch cells were used in cell transplantation therapies.

In a fourth method, ES cells can be isolated from the primordial germcells found in the genital ridges of post-implanted embryos.

As described above, all presently available methods depend oncontroversial sources—embryos (either created naturally or via cloning),fetal tissue and via the mixing of materials of multiple species. Thecontroversy surrounding the sources for such cells, according to manyleading scientists and public and private organizations including theNIH, has greatly compromised and slowed the study of such cells andtheir application.

There is thus a great demand for alternative methods of generatingpluripotent cells.

SUMMARY OF THE INVENTION

The present invention provides engineered somatic cells, in which one ormore endogenous pluripotency gene(s) is operably linked to a selectablemarker in such a manner that the expression of the selectable markersubstantially matches the expression of the endogenous pluripotency geneto which the marker is linked. The invention also provides transgenicmice containing these engineered somatic cells.

The present invention also provides methods for reprogramming somaticcells to a less differentiated state. In the methods, engineered somaticcells of the invention are treated with an agent. Cells that express theselectable marker are then selected, and assessed for pluripotencycharacteristics. The treatment with an agent may be contacting the cellswith an agent which alters chromatin structure, or may be transfectingthe cells with at least one pluripotency gene, or both.

The present invention further provides methods for identifying an agentthat reprograms somatic cells to a less differentiated state. In themethods, the engineered somatic cells described above are contacted witha candidate agent. Cells that express the selectable marker are thenselected, and assessed for pluripotency characteristics. The presence ofat least a subset of pluripotency characteristics indicates that theagent is capable of reprogramming somatic cells to a less-differentiatedstate. The agents identified by the present invention can then by usedto reprogram somatic cells by contacting somatic cells with the agents.

The present invention also provides methods for identifying a gene thatcauses the expression of at least one endogenous pluripotency gene insomatic cells. In the methods, the engineered somatic cells aretransfected with a cDNA library prepared from a pluripotent cell, suchas an ES cell. The cells that express the appropriate selectable markerare then selected, and the expression of the appropriate endogenouspluripotency gene is examined. The expression of an endogenouspluripotency gene indicates that the cDNA encodes a protein whoseexpression in the cell results in, directly or indirectly, expression ofthe endogenous pluripotency gene.

The present invention further provides methods for treating a conditionin an individual in need of such treatment. In certain embodiments,somatic cells are obtained from the individual and reprogrammed by themethods of the invention under conditions suitable for the cells todevelop into cells of a desired cell type. The reprogrammed cells of adesired cell type are then harvested and introduced into the individualto treat the condition. In certain further embodiments, the somaticcells obtained from the individual contains a mutation in one or moregenes. In these instances, the methods are modified so that the somaticcells obtained from the individual are first treated to restore the oneor more normal gene(s) to the cells such that the resulting cells carrythe normal endogenous gene, which are then introduced into theindividual. In certain other embodiments, methods of the invention canbe used to treat individuals in need of a functional organ. In themethods, somatic cells are obtained from an individual in need of afunctional organ, and reprogrammed by the methods of the invention toproduce reprogrammed somatic cells. Such reprogrammed somatic cells arethen cultured under conditions suitable for development of thereprogrammed somatic cells into a desired organ, which is thenintroduced into the individual. The methods are useful for treating anyone of the following conditions: a neurological, endocrine, structural,skeletal, vascular, urinary, digestive, integumentary, blood,autoimmune, inflammatory, or muscular condition.

The present invention also provides methods for producing a clonedanimal. In the methods, a somatic cell is isolated from an animal havingdesired characteristics, and reprogrammed using the methods of theinvention to produce one or more reprogrammed pluripotent somatic cell(“RPSC”). The RPSCs are then inserted into a recipient embryo, and theresulting embryo is cultured to produce an embryo of suitable size forimplantation into a recipient female, which is then transferred into arecipient female to produce a pregnant female. The pregnant female ismaintained under conditions appropriate for carrying the embryo to termto produce chimeric animal progeny, which is then bred with a wild typeanimal to produce a cloned animal.

In certain embodiments, the RPSCs may alternatively be cryopreserved forfuture cloning uses. In certain other embodiments, genetic modification,such as a targeted mutation, may be introduced into the RPSCs prior toits insertion into a recipient embryo.

The present invention also provides methods for producing a clonedavian. In the methods, a somatic cell is isolated from an avian havingdesired characteristics, and reprogrammed using the methods of theinvention to produce one or more reprogrammed pluripotent somatic cell(“RPSC”). The RPSCs are then inserted into eggs that are unable todevelop into an embryo, and the resulting eggs are then incubated toproduce avian offspring having the genotype of the RPSC, therebyproducing a cloned avian.

It is contemplated that all embodiments described above are applicableto all different aspects of the invention. It is also contemplated thatany of the above embodiments can be freely combined with one or moreother such embodiments whenever appropriate.

Specific embodiments of the invention are described in more detailbelow. However, these are illustrative embodiments, and should not beconstrued as limiting in any respect.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts an inducible Oct4 allele.

FIG. 2 shows the expression of the inducible Oct4 transgene by Northernblot and Western blot analysis.

DETAILED DESCRIPTION OF THE INVENTION Overview

Presently, human ES cells or ES-like cells can only be generated fromcontroversial sources. It would be useful to reprogram somatic cellsdirectly into pluripotent cells. Nuclei from somatic cells retain thetotopotency potential to direct development of an animal, asdemonstrated by nuclear transfer technology. It would be useful toreprogram somatic cells directly into ES cells without the use ofoocytes and nuclear transfer technology.

Applicants have devised novel methods of reprogramming somatic cells togenerate pluripotent cells or multipotent cells. Applicants have alsodevised novel methods to identify agents that reprogram somatic cells.The methods take advantage of the engineered somatic cells designed byApplicants, in which an endogenous gene typically associated withpluripotency (“pluripotency gene”) is engineered to be operably linkedto a selectable marker in a manner that the expression the endogenouspluripotency gene substantially matches the expression of the selectablemarker. Because pluripotency genes are generally expressed only inpluripotent cells and not in somatic cells, the expression of anendogenous pluripotent gene(s) is an indication of successfulreprogramming. Having a selectable marker operably linked to anendogenous pluripotency gene gives one a powerful mechanism to selectfor potentially reprogrammed somatic cells, which likely is a rareoccurrence. The resulting cells may be further assessed for pluripotencycharacteristics to confirm whether a somatic cell has been successfullyreprogrammed to pluripotency.

Generating pluripotent or multipotent cells by somatic cellreprogramming using the methods of the present invention has at leasttwo advantages. First, the methods of the present invention allow one togenerate autologous pluripotent cells, which are cells specific to apatient. The use of autologous cells in cell therapy offers a majoradvantage over the use of non-autologous cells, which are likely to besubject to immunological rejection. In contrast, autologous cells areunlikely to elicit significant immunological responses (See Munsie etal, 2000). Second, the methods of the present invention allow one togenerate pluipotent without using embryos, oocytes and/or nucleartransfer technology.

A pluripotent cell is a cell that has the potential to divide in vitrofor a long period of time (greater than one year) and has the uniqueability to differentiate into cells derived from all three embryonicgerm layers—endoderm, mesoderm and ectoderm.

A multipotent cell is a cell that is able to differentiate into some butnot all of the cells derived from all three germ layers. Thus, amultipotent cell is a partially differentiated cell. Adult stem cellsare multipotent cells. Known adult stem cells include, for example,hematopoietic stem cells and neural stem cells. A hematopoietic stemcell is multipotent because it has the ability to differentiate into alltypes of specific blood cells, but it is unlikely that they candifferentiate into all cells of a given animal or human.Multipotent/adult stem cells have a great deal of promise in researchand in the area of therapeutic applications. For example,multipotent/adult stem cells have already been used in humans inattempts to treat certain blood, neural and cancer diseases.

The term “pluripotency gene”, as used herein, refers to a gene that isassociated with pluripotency. The expression of a pluripotency gene istypically restricted to pluripotent stem cells, and is crucial for thefunctional identity of pluripotent stem cells. The transcription factorOct-4 (also called Pou5fl, Oct-3, Oct3/4) is an example of apluripotency gene. Oct-4 has been shown to be required for establishingand maintaining the undifferentiated phenotype of ES cells and plays amajor role in determining early events in embryogenesis andcellular-differentiation (Nichols et al., 1998, Cell 95:379-391; Niwa etal., 2000, Nature Genet. 24:372-376). Oct-4 is down-regulated as stemcells differentiate into specialised cells. Other exemplary pluripotencygenes include Nanog, and Stella (See Chambers et al., 2003, Cell 113:643-655; Mitsui et al., Cell. 2003, 113(5):631-42; Bortvin et al.Development. 2003, 130(8):1673-80; Saitou et al., Nature. 2002, 418(6895):293-300.

Engineered Somatic Cells and Transgenic Mice Comprising Such Cells

The present invention provides somatic cells comprising an endogenouspluripotency gene linked to DNA encoding a selectable marker in such amanner that the expression of the selectable marker substantiallymatches the expression of the endogenous pluripotency gene. In oneembodiment, the somatic cells of the present invention comprise a firstendogenous pluripotency gene linked to DNA encoding a first selectablemarker in such a manner that the expression of the first selectablemarker substantially matches the expression of the first endogenouspluripotency gene. The somatic cells may also be engineered to compriseany number of endogenous pluripotency genes respectively linked to adistinct selectable marker. Thus, in another embodiment, the somaticcells of the present invention comprise two endogenous pluripotencygenes, each of which is linked to DNA encoding a distinct selectablemarker. In a further embodiment, the somatic cells of the presentinvention comprise three endogenous pluripotency genes, each of which islinked to DNA encoding a distinct selectable marker. The somatic cellsdescribed above will be collectively referred in this application as“engineered somatic cells.” The engineered somatic cells may be furtherengineered to have one or more pluripotency gene expressed as atransgene under an inducible promoter.

The selectable marker is linked to an appropriate endogenouspluripotency gene such that the expression of the selectable markersubstantially matches the expression of the endogenous pluripotencygene. By “substantially match”, it is meant that the expression of theselectable marker substantially reflects the expression pattern of theendogenous pluripotency gene. In other words, the selectable marker andthe endogenous pluripotency gene are co-expressed. For purpose of thepresent invention, it is not necessary that the expression level of theendogenous gene and the selectable marker is the same or even similar.It is only necessary that the cells in which an endogenous pluripotencygene is activated will also express the selectable marker at a levelsufficient to confer a selectable phenotype on the reprogrammed cells.For example, when the selectable marker is a marker that confersresistance to a lethal drug (a “drug resistance marker”), the cells areengineered in a way that allows cells in which an endogeneouspluripotency gene is activated to also express the drug resistancemarker at a sufficient level to confer on reprogrammed cells resistanceto lethal drugs. Thus, reprogrammed cells will survive and proliferatewhereas non-reprogrammed cells will die.

The DNA encoding a selectable marker may be inserted downstream from theend of the open reading frame (ORF) encoding the desired endogenouspluripotency gene, anywhere between the last nucleotide of the ORF andthe first nucleotide of the polyadenylation site. An internal ribosomeentry site (IRES) may be placed in front of the DNA encoding theselectable marker. Alternatively, the DNA encoding a selectable markermay be inserted anywhere within the ORF of the desired endogenouspluripotency gene, downstream of the promoter, with a terminationsignal. An internal ribosome entry site (IRES) may be placed in front ofthe DNA encoding the selectable marker. The selectable marker may beinserted into only one allele, or both alleles, of the endogenouspluripotency gene.

The somatic cells in the invention may be primary cells or immortalizedcells. Such cells may be primary cells (non-immortalized cells), such asthose freshly isolated from an animal, or may be derived from a cellline (immortalized cells).

The somatic cells in the present invention are mammalian cells, such as,for example, human cells or mouse cells. They may be obtained bywell-known methods, from different organs, e.g., skin, lung, pancreas,liver, stomach, intestine, heart, reproductive organs, bladder, kidney,urethra and other urinary organs, etc., generally from any organ ortissue containing live somatic cells. Mammalian somatic cells useful inthe present invention include, by way of example, adult stem cells,sertoli cells, endothelial cells, granulosa epithelial, neurons,pancreatic islet cells, epidermal cells, epithelial cells, hepatocytes,hair follicle cells, keratinocytes, hematopoietic cells, melanocytes,chondrocytes, lymphocytes (B and T lymphocytes), erythrocytes,macrophages, monocytes, mononuclear cells, fibroblasts, cardiac musclecells, and other muscle cells, etc. generally any live somatic cells.The term “somatic cells”, as used herein, also includes adult stemcells. An adult stem cell is a cell that is capable of giving rise toall cell types of a particular tissue. Exemplary adult stem cellsinclude hematopoietic stem cells, neural stem cells, and mesenchymalstem cells.

In one embodiment, the engineered somatic cells are obtained from atransgenic mouse comprising such engineered somatic cells. Suchtransgenic mouse can be produced using standard techniques known in theart. For example, Bronson et al. describe a technique for inserting asingle copy of a transgene into a chosen chromosomal site. See Bronsonet al., 1996. Briefly, a vector containing the desired integrationconstruct (for example, a construct containing a selectable markerlinked to a pluripotency gene) is introduced into ES cells by standardtechniques known in the art. The resulting ES cells are screened for thedesired integration event, in which the knock-in vector is integratedinto the desired endogenous pluripotency gene locus such that theselectable marker is integrated into the genomic locus of thepluripotency gene and is under the control of the pluripotency genepromoter. The desired ES cell is then used to produce transgenic mousein which all cell types contain the correct integration event. Desiredtypes of cells may be selectively obtained from the transgenic mouse andmaintained in vitro. In one embodiment, two or more transgenic mice maybe created, each carrying a distinct integration construct. These micemay then be bred to generate mice that carry multiple desiredintegration construct. For example, one type of transgenic mouse may becreated to carry an endogenous pluripotency gene linked to a selectablemarker, while a second type of transgenic mouse may be created to carrya pluripotency gene expressed as a transgene under an induciblepromoter. These two types of mice may then be bred to generatetransgenic mice that have both a selectable marker linked to anendogenous pluripotency gene and an additional pluripotency geneexpressed as a transgene under an inducible promoter. These twopluripotency genes may or may not be the same. Many variables arecontemplated: the identity of the endogenous pluripotency gene linked tomarker, the identity of the pluripotency gene expressed as a transgene,and the number of the endogenous pluripotency gene linked to aselectable marker, and the number of pluripotency gene expressed as atransgene. The present invention encompasses all possible combinationsof these variables.

Alternatively, engineered somatic cells of the present invention may beproduced by direct introduction of the desired construct into somaticcells. DNA construct may be introduced into cells by any standardtechnique known in the art, such as viral transfection (eg. using anadenoviral system) or liposome-mediated transfection. Any means known inthe art to generate somatic cells with targeted integration can be usedto produce somatic cells of the invention. In mammalian cells,homologous recombination occurs at much lower frequency compared tonon-homologous recombination. To facilitate the selection of homologousrecombination events over the non-homologous recombination events, atleast two enrichment methods have been developed: the positive-negativeselection (PNS) method and the “promoterless” selection method (Sedivyand Dutriaux, 1999). Briefly, PNS, the first method, is in genetic termsa negative selection: it selects against recombination at the incorrect(non-homologous) loci by relying on the use of a negatively selectablegene that is placed on the flanks of a targeting vector. On the otherhand, the second method, the “promoterless” selection, is a positiveselection in genetic terms: it selects for recombination at the correct(homologous) locus by relying on the use of a positively selectable genewhose expression is made conditional on recombination at the homologoustarget site. The disclosure of Sedivy and Dutriaux is incorporatedherein.

A selectable marker, as used herein, is a marker that, when expressed,confers upon recipient cells a selectable phenotype, such as antibioticresistance, resistance to a cytotoxic agent, nutritional prototrophy orexpression of a surface protein. The presence of a selectable markerlinked to an endogenous pluripotency gene makes it possible to identifyand select reprogrammed cells in which the endogenous pluripotency geneis expressed. A variety of selectable marker genes can be used, such asneomycin resistance gene (neo), puromycin resistance gene (puro),guanine phosphoribosyl transferase (gpt), dihydrofolate reductase(DHFR), adenosine deaminase (ada), puromycin-N-acetyltransferase (PAC),hygromycin resistance gene (hyg), multidrug resistance gene (mdr), andhisD gene.

The present invention further provides transgenic mice comprising thesomatic cells of the invention.

Methods for Reprogramming Somatic Cells

The present invention further provides methods for reprogramming somaticcells to a less differentiated state. The resulting cells are termed“reprogrammed somatic cells” (“RSC”) herein. A RSC may be a reprogrammedpluripotent somatic cell (“RPSC”), a reprogrammed multipotent somaticcell (“RMSC”), or a reprogrammed somatic cell of varying differentiationstatus.

In general, the methods comprise treating the engineered somatic cellswith an agent. The treatment with an agent may be contacting the cellswith an agent which alters chromatin structure, or may be transfectingthe cells with one or more pluripotency gene, or both. The above twotreatments may be concurrent, or may be sequential, with no particularpreference for order. In a further embodiment, reprogrammed somaticcells are identified by selecting for cells that express the appropriateselectable marker. In still a further embodiment, reprogrammed somaticcells are further assessed for pluripotency characteristics. Thepresence of pluripotency characteristics indicates that the somaticcells have been reprogrammed to a pluripotent state.

Differentiation status of cells is a continuous spectrum, withterminally differentiated state at one end of this spectrum andde-differentiated state (pluripotent state) at the other end.Reprogramming, as used herein, refers to a process that alters orreverses the differentiation status of a somatic cell, which can beeither partially or terminally differentiated. Reprogramming includescomplete reversion, as well as partial reversion, of the differentiationstatus of a somatic cell. In other words, the term “reprogramming”, asused herein, encompasses any movement of the differentiation status of acell along the spectrum toward a less-differentiated state. For example,reprogramming includes reversing a multipotent cell back to apluripotent cell, reversing a terminally differentiated cell back toeither a multipotent cell or a pluripotent cell. In one embodiment,reprogramming of a somatic cell turns the somatic cell all the way backto a pluripotent state. In another embodiment, reprogramming of asomatic cell turns the somatic cell back to a multipotent state. Theterm “less-differentiated state”, as used herein, is thus a relativeterm and includes a completely de-differentiated state and a partiallydifferentiated state.

The term “pluripotency characteristics”, as used herein, refers to manycharacteristics associated with pluripotency, including, for example,the ability to differentiate into all types of cells and an expressionpattern distinct for a pluripotent cell, including expression ofpluripotency genes, expression of other ES cell markers, and on a globallevel, a distinct expression profile known as “stem cell molecularsignature” or “stemness.”

Thus, to assess reprogrammed somatic cells for pluripotencycharacteristics, one may analyze such cells for different growthcharacteristics and ES cell-like morphology. Cells may be injectedsubcutaneously into immunocompromised SCID mice to induce teratomas (astandard assay for ES cells). ES-like cells can be differentiated intoembryoid bodies (another ES specific feature). Moreover, ES-like cellscan be differentiated in vitro by adding certain growth factors known todrive differentiation into specific cell types. Self-renewing capacity,marked by induction of telomerase activity, is another plutipotencycharacteristics that can be monitored. One may carry out functionalassays of the reprogrammed somatic cells by introducing them intoblastocysts and determine whether the cells are capable of giving riseto all cell types. See Hogan et al., 2003. If the reprogrammed cells arecapable of forming a few cell types of the body, they are multipotent;if the reprogrammed cells are capable of forming all cell types of thebody including germ cells, they are pluripotent.

One may also examine the expression of an individual pluripotency genein the reprogrammed somatic cells to assess their pluripotencycharacteristics. Additionally, one may assess the expression of other EScell markers. Stage-specific embryonic 15 antigens-1, -3, and -4(SSEA-1, SSEA-3, SSEA-4) are glycoproteins specifically expressed inearly embryonic development and are markers for ES cells (Solter andKnowles, 1978, Proc. Natl. Acad. Sci. USA 75:5565-5569; Kannagi et al.,1983, EMBO J 2:2355-2361). Elevated expression of the enzyme AlkalinePhosphatase (AP) is another marker associated with undifferentiatedembryonic stem cells (Wobus et al., 1984, Exp. Cell 152:212-219; Peaseet al., 1990, Dev. Biol. 141:322-352). Other stem/progenitor cellsmarkers include the intermediate neurofilament nestin (Lendahl et al.,1990, Cell 60:585-595; Dah-Istrand et al., 1992, J.

Cell Sci. 103:589-597), the membrane glycoprotein prominin/AC133(Weigmann et al., 1997, Proc. Natl. Acad. USA 94:12425-12430; Corbeil etal., 1998, Blood 91:2625-22626), the transcription factor Tcf-4 (Korineket al, 1998, Nat. Genet. 19: 379-383; Lee et al., 1999, J. Biol. Chem.274.1 566-1 572), and the transcription factor Cdx1 (Duprey et al.,1988, Genes Dev. 2:1647-1654; Subramania'n et al., 1998, Differentiation64:11-1 8).

One may additionally conduct expression profiling of the reprogrammedsomatic cells to assess their pluripotency characteristics. Pluripotentcells, such as embryonic stem cells, and multipotent cells, such asadult stem cells, are known to have a distinct pattern of global geneexpression profile. This distinct pattern is termed “stem cell molecularsignature”, or “stemness”. See, for example, Ramalho-Santos et al.,Science 298: 597-600 (2002); Ivanova et al., Science 298: 601-604.

Somatic cells may be reprogrammed to gain either a complete set of thepluripotency characteristics and are thus pluripotent. Alternatively,somatic cells may be reprogrammed to gain only a subset of thepluripotency characteristics. In another alternative, somatic cells maybe reprogrammed to be multipotent.

In a further embodiment, in conjunction with contacting the somaticcells of the invention with an agent which alters chromatin structure,at least one gene that affects pluripotent state of a cell may befurther introduced into the same cells. This may be carried outsequentially. For example, the somatic cells of the invention may befirst contacted with an agent which alters chromatin structure. Then atleast one pluripotency gene can be introduced into the same cells, orvice versa. Alternatively, the two steps may be carried outsimultaneously.

Genes that affect pluripotent state of a cell includes pluripotencygenes, genes involved in chromatin remodeling, and genes that areimportant for maintaining pluripotency, such as LIF, BMP, and PD098059(See Cell, 115: 281-292 (2003); Philos Trans R Soc Lond B Biol Sci. 2003Aug. 29; 358(1436):1397-402).

The exogenously introduced pluripotency gene may be carried out inseveral ways. In one embodiment, the exogenously introduced pluripotencygene may be expressed from a chromosomal locus different from theendogenous chromosomal locus of the pluripotency gene. Such chromosomallocus may be a locus with open chromatin structure, and contain gene(s)dispensible for a somatic cell. In other words, the desirablechromosomal locus contains gene(s) whose disruption will not cause cellsto die. Exemplary chromosomal loci include, for example, the mouse ROSA26 locus and type II collagen (Col2a1) locus (See Zambrowicz et al.,1997) The exogenously introduced pluripotency gene may be expressed froman inducible promoter such that their expression can be regulated asdesired.

In an alternative embodiment, the exogenously introduced pluripotencygene may be transiently transfected into cells, either individually oras part of a cDNA expression library, prepared from pluripotent cells.Such pluripotent cells may be embryonic stem cells, oocytes,blastomeres, inner cell mass cells, embryonic germ cells, embryoid body(embryonic) cells, morula-derived cells, teratoma (teratocarcinoma)cells, and multipotent partially differentiated embryonic stem cellstaken from later in the embryonic development process.

The cDNA library is prepared by conventional techniques. Briefly, mRNAis isolated from an organism of interest. An RNA-directed DNA polymeraseis employed for first strand synthesis using the mRNA as template.Second strand synthesis is carried out using a DNA-directed DNApolymerase which results in the cDNA product. Following conventionalprocessing to facilitate cloning of the cDNA, the cDNA is inserted intoan expression vector such that the cDNA is operably linked to at leastone regulatory sequence. The choice of expression vectors for use inconnection with the cDNA library is not limited to a particular vector.Any expression vector suitable for use in mouse cells is appropriate. Inone embodiment, the promoter which drives expression from the cDNAexpression construct is an inducible promoter. The term regulatorysequence includes promoters, enhancers and other expression controlelements. Exemplary regulatory sequences are described in Goeddel; GeneExpression Technology: Methods in Enzymology, Academic Press, San Diego,Calif. (1990). For instance, any of a wide variety of expression controlsequences that control the expression of a DNA sequence when operativelylinked to it may be used in these vectors to express cDNAs. Such usefulexpression control sequences, include, for example, the early and latepromoters of SV40, tet promoter, adenovirus or cytomegalovirus immediateearly promoter, the lac system, the trp system, the TAC or TRC system,T7 promoter whose expression is directed by T7 RNA polymerase, the majoroperator and promoter regions of phage lambda, the control regions forfd coat protein, the promoter for 3-phosphoglycerate kinase or otherglycolytic enzymes the promoters of acid phosphatase, e.g., Pho5, thepromoters of the yeast α-mating factors, the polyhedron promoter of thebaculovirus system and other sequences known to control the expressionof genes of prokaryotic or eukaryotic cells or their viruses, andvarious combinations thereof. It should be understood that the design ofthe expression vector may depend on such factors as the choice of thehost cell to be transformed and/or the type of protein desired to beexpressed. Moreover, the vector's copy number, the ability to controlthat copy number and the expression of any other protein encoded by thevector, such as antibiotic markers, should also be considered.

The exogenously introduced pluripotency gene may be expressed from aninducible promoter. The term “inducible promoter”, as used herein,refers to a promoter that, in the absence of an inducer (such as achemical and/or biological agent), does not direct expression, ordirects low levels of expression of an operably linked gene (includingcDNA), and, in response to an inducer, its ability to direct expressionis enhanced. Exemplary inducible promoters include, for example,promoters that respond to heavy metals (CRC Boca Raton, Fla. (1991),167-220; Brinster et al. Nature (1982), 296, 39-42), to thermal shocks,to hormones (Lee et al. P.N.A.S. USA (1988), 85, 1204-1208; (1981), 294,228-232; Klock et al. Nature (1987), 329, 734-736; Israel and Kaufman,Nucleic Acids Res. (1989), 17, 2589-2604), promoters that respond tochemical agents, such as glucose, lactose, galactose or antibiotic.

A tetracycline-inducible promoter is an example of an inducible promoterthat responds to an antibiotics. See Gossen et al., 2003. Thetetracycline-inducible promoter comprises a minimal promoter linkedoperably to one or more tetracycline operator(s). The presence oftetracycline or one of its analogues leads to the binding of atranscription activator to the tetracycline operator sequences, whichactivates the minimal promoter and hence the transcription of theassociated cDNA. Tetracycline analogue includes any compound thatdisplays structural homologies with tetracycline and is capable ofactivating a tetracycline-inducible promoter. Exemplary tetracyclineanalogues includes, for example, doxycycline, chlorotetracycline andanhydrotetracycline.

Thus, in one embodiment, the present invention provides mice and somaticcells carrying at least on pluripotency gene expressed as a transgeneunder an inducible promoter. It is possible that somatic cells with suchinducible pluripotency transgene(s) are more prone to be reprogrammed.

Any of the engineered somatic cells of the present invention may be usedin the methods. In one embodiment, somatic cells used in the methodscomprise only one endogenous pluripotency gene linked to a firstselectable marker, and the selection step is carried out to select forthe expression of the first selectable marker. In an alternativeembodiment, the somatic cells used in the methods comprise any number ofendogenous pluripotency genes, each of which is linked to a distinctselectable marker respectively, and the selection step is carried out toselect for at least a subset of the selectable markers. For example, theselection step may be carried out to select for all the selectablemarkers linked to the various endogenous pluripotency genes.

In an alternative embodiment, somatic cells used in the method comprisea selectable marker linked to an endogenous pluripotency gene and anadditional pluripotency gene expressed as a transgene under an induciblepromoter. For these cells, the method of reprogramming may comprisesinduce the expression of the pluripotency transgene and select for theexpression of the selectable marker. The method may further comprisecontacting the somatic cells with an agent that alter chromaticstructure.

Without wishing to be bound by theory, the agents used in the method maycause chromatin to take on a more open structure, which is morepermissive for gene expression. DNA methylation and histone acetylationare two known events that alter chromatin toward a more closedstructure. For example, loss of methylation by genetic deletion of DNAmethylation enzyme Dnmt1 in fibroblasts results in reactivation ofendogenous Oct4 gene. See J. Biol. Chem. 277: 34521-30, 2002; andBergman and Mostoslavsky, Biol. Chem. 1990. Thus, DNA methylationinhibitors and histone deacetyation inhibitors are two classes of agentsthat may be used in the methods of the invention. Exemplary agentsinclude 5-aza-cytidine, TSA and valproic acid.

In another embodiment, methods of the invention may further includerepeating the steps of treating the cells with an agent. The agent usedin the repeating treatment may be the same as, or different from, theone used during the first treatment.

Methods for Screening for an Agent that Reprograms Somatic Cells

The present invention also provides methods for identifying an agentthat reprograms somatic cells to a less-differentiated state, as well asthe agents thus identified. In one embodiment, the methods comprisecontacting the engineered somatic cells of the invention with acandidate agent, selecting for cells that express the appropriateselectable marker. The presence of cells that express the appropriateselectable marker indicates that the agent reprograms somatic cells.Such an agent is referred as a “reprogramming agent” for purpose of thisapplication.

In a further embodiment, the methods comprise contacting the engineeredsomatic cells of the invention with a candidate agent, selecting forcells that express the appropriate selectable marker, and assessing thecells so selected for pluripotency characteristics. The presence of acomplete set of pluripotency characteristics indicates that the agentreprograms somatic cells to become pluripotent.

Candidate agents used in the invention encompass numerous chemicalclasses, though typically they are organic molecules, including smallorganic compounds. Candidate agents are also found among biomoleculesincluding peptides, saccharides, fatty acids, steroids, purines,pyrimidines, nucleic acids and derivatives, structural analogs orcombinations thereof.

Candidate agents may be naturally arising, recombinant or designed inthe laboratory. The candidate agents may be isolated frommicroorganisms, animals, or plants, or may be produced recombinantly, orsynthesized by chemical methods known in the art. In some embodiments,candidate agents are isolated from libraries of synthetic or naturalcompounds using the methods of the present invention. For example,numerous means are available for random and directed synthesis of a widevariety of organic compounds and biomolecules, including expression ofrandomized oligonucleotides and oligopeptides. Alternatively, librariesof natural compounds in the form of bacterial, fungal, plant and animalextracts are available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means, and maybe used to produce combinatorial libraries. Known pharmacological agentsmay be subjected to directed or random chemical modifications, includingacylation, alkylation, esterification, amidification, to producestructural analogs.

There are numerous commercially available compound libraries, including,for example, the Chembridge DIVERSet. Libraries are also available fromacademic investigators, such as the Diversity set from the NCIdevelopmental therapeutics program.

The screening methods mentioned above are based on assays performed oncells. These cell-based assays may be performed in a high throughputscreening (HTS) format, which has been described in the art. Forexample, Stockwell et al. described a high-throughput screening of smallmolecules in miniaturized mammalian cell-based assays involvingpost-translational modifications (Stockwell et al., 1999). Likewise,Qian et al. described a leukemia cell-based assay for high-throughtputscreening for anti-cancer agents (Qian et al., 2001). Both referencesare incorporated herein in their entirety.

A reprogramming agent may belong to any one of many differentcategories. For example, a reprogramming agent may be a chromatinremodeling agent. A chromatin remodeling agent may be a protein involvedin chromatin remodeling or an agent known to alter chromatin toward amore open structure, such as a DNA methylation inhibitor or a histonedeacetyation inhibitor. Exemplary compounds include 5-aza-cytidine, TSAand valproic acid. For another example, such an agent may be apluripotency protein, including, for example, Nanog, Oct-4 and Stella.Such an agent may also be a gene essential for pluripotency, including,for example, Sox2, FoxD3, and LIF, and Stat3. See Smith et al. 1988,William et al., 1988, Ihle, 1996, Avilion et al., 2003, and Hanna etal., 2002)

Methods for Reprogramming Somatic Cells with a Reprogramming Agent

The reprogramming agent identified by the methods of the presentinvention is useful for reprogramming somatic cells into pluripotent ormultipotent cells. Accordingly, the present invention provides methodsfor reprogramming somatic cells to a less differentiated state,comprising contacting somatic cells with a reprogramming agent. Thesomatic cells used may be native somatic cells, or engineered somaticcells. It is not necessary for these cells to carry a selectable markerintegrated into the endogenous locus of a pluripotency gene.

Reprogrammed Somatic Cells and These Uses

The present invention also provides reprogrammed somatic cells (RSCs),including reprogrammed pluripotent somatic cells (RPSCs), produced bythe methods of the invention. These methods, useful for the generationof cells of a desired cell type, have wide range of applications. Forone example, these methods have applications in livestock management,involving the precise genetic manipulation of animals for economic orhealth purposes. For another example, these methods have medicalapplication in treating or preventing a condition.

Accordingly, the invention provides methods for the treatment orprevention of a condition in a mammal. In one embodiment, the methodsstart with obtaining somatic cells from the individual, reprogrammingthe somatic cells so obtained by methods of the present invention toobtain RPSCs. The RPSCs are then cultured under conditions suitable fordevelopment of the RPSCs into cells of a desired cell type. Thedeveloped cells of the desired cell type are harvested and introducedinto the individual to treat the condition. In an alternativeembodiment, the methods start with obtaining somatic cells from theindividual, reprogramming the somatic cells so obtained by methods ofthe present invention. The RPSCs are then cultured under conditionssuitable for development of the RPSCs into a desired organ, which isharvested and introduced into the individual to treat the condition.

The RPSCs of the present invention are ES-like cells, and thus may beinduced to differentiate to obtain the desired cell types according toknown methods to differentiate ES cells. For example, the RPSCs may beinduced to differentiate into hematopoietic stem cells, muscle cells,cardiac muscle cells, liver cells, cartilage cells, epithelial cells,urinary tract cells, etc., by culturing such cells in differentiationmedium and under conditions which provide for cell differentiation.Medium and methods which result in the differentiation of embryonic stemcells are known in the art as are suitable culturing conditions.

For example, Palacios et al., Proc. Natl. Acad. Sci., USA, 92: 7530-37(1995) teaches the production of hematopoietic stem cells from anembryonic cell line by subjecting stem cells to an induction procedurecomprising initially culturing aggregates of such cells in a suspensionculture medium lacking retinoic acid followed by culturing in the samemedium containing retinoic acid, followed by transferral of cellaggregates to a substrate which provides for cell attachment.

Moreover, Pedersen, J. Reprod. Fertil. Dev., 6: 543-52 (1994) is areview article which references numerous articles disclosing methods forin vitro differentiation of embryonic stem cells to produce variousdifferentiated cell types including hematopoietic cells, muscle, cardiacmuscle, nerve cells, among others.

Further, Bain et al., Dev. Biol., 168:342-357 (1995) teaches in vitrodifferentiation of embryonic stem cells to produce neural cells whichpossess neuronal properties. These references are exemplary of reportedmethods for obtaining differentiated cells from embryonic or stem-likecells. These references and in particular the disclosures thereinrelating to methods for differentiating embryonic stem cells areincorporated by reference in their entirety herein.

Thus, using known methods and culture medium, one skilled in the art mayculture the subject embryonic or stem-like cells to obtain desireddifferentiated cell

types, e.g., neural cells, muscle cells, hematopoietic cells, etc. Inaddition, the use of inducible Bcl-2 or Bcl-x1 might be useful forenhancing in vitro development of specific cell lineages. In vivo, BcI-2prevents many, but not all, forms of apoptotic cell death that occurduring lymphoid and neural development. A thorough discussion of howBcl-2 expression might be used to inhibit apoptosis of relevant celllineages following transfection of donor cells is disclosed in U.S. Pat.No. 5,646,008, which is herein incorporated by reference.

The subject RPSCs may be used to obtain any desired differentiated celltype. Therapeutic usages of such differentiated human cells areunparalleled. For example, human hematopoietic stem cells may be used inmedical treatments requiring bone marrow transplantation. Suchprocedures are used to treat many diseases, e.g., late stage cancerssuch as ovarian cancer and leukemia, as well as diseases that compromisethe immune system, such as AIDS. Hematopoietic stem cells can beobtained, e.g., by fusing adult somatic cells of a cancer or AIDSpatient, e.g., epithelial cells or lymphocytes with an enucleatedoocyte, e.g., bovine oocyte, obtaining embryonic or stem-like cells asdescribed above, and culturing such cells under conditions which favordifferentiation, until hematopoietic stem cells are obtained. Suchhematopoietic cells may be used in the treatment of diseases includingcancer and AIDS.

The methods of the present invention can also be used to treat, prevent,or stabilize a neurological disease such as Alzheimer's disease,Parkinson's disease, Huntington's disease, or ALS, lysosomal storagediseases, multiple sclerosis, or a spinal cord injury. For example,somatic cells may be obtained from the individual in need of treatment,and reprogrammed to gain pluripotency, and cultured to deriveneurectoderm cells that may be used to replace or assist the normalfunction of diseased or damaged tissue.

For the treatment or prevention of endocrine conditions, RPSCs thatproduce a hormone, such as a growth factor, thyroid hormone,thyroid-stimulating hormone, parathyroid hormone, steroid, serotonin,epinephrine, or norepinephrine may be administered to a mammal.Additionally, reprogrammed epithelial cells may be administered torepair damage to the lining of a body cavity or organ, such as a lung,gut, exocrine gland, or urogenital tract. It is also contemplated thatRPSCs may be administered to a mammal to treat damage or deficiency ofcells in an organ such as the bladder, brain, esophagus, fallopian tube,heart, intestines, gallbladder, kidney, liver, lung, ovaries, pancreas,prostate, spinal cord, spleen, stomach, testes, thymus, thyroid,trachea, ureter, urethra, or uterus.

The great advantage of the present invention is that it provides anessentially limitless supply of isogenic or synegenic human cellssuitable for transplantation. Therefore, it will obviate the significantproblem associated with current transplantation methods, i.e., rejectionof the transplanted tissue which may occur because of host versus graftor graft versus host rejection. Conventionally, rejection is preventedor reduced by the administration of anti-rejection drugs such ascyclosporin. However, such drugs have significant adverse side-effects,e.g., immunosuppression, carcinogenic properties, as well as being veryexpensive. The present invention should eliminate, or at least greatlyreduce, the need for anti-rejection drugs, such as cyclosporine, imulan,FK-506, glucocorticoids, and rapamycin, and derivatives thereof.

RPSCs may also be combined with a matrix to form a tissue or organ invitro or in vivo that may be used to repair or replace a tissue or organin a recipient mammal. For example, RPSCs may be cultured in vitro inthe presence of a matrix to produce a tissue or organ of the urogenitalsystem, such as the bladder, clitoris, corpus cavermosum, kidney,testis, ureter, uretal valve, or urethra, which may then be transplantedinto a mammal (Atala, Curr. Opin. Urol. 9(6):517-526, 1999). In anothertransplant application, synthetic blood vessels are formed in vitro byculturing reprogrammed cells in the presence of an appropriate matrix,and then the vessels are transplanted into a mammal for the treatment orprevention of a cardiovascular or circulatory condition. For thegeneration of donor cartilage or bone tissue, RPSCs such as chondrocytesor osteocytes are cultured in vitro in the presence of a matrix underconditions that allow the formation of cartilage or bone, and then thematrix containing the donor tissue is administered to a mammal.Alternatively, a mixture of the cells and a matrix may be administeredto a mammal for the formation of the desired tissue in vivo. Preferably,the cells are attached to the surface of the matrix or encapsulated bythe matrix. Examples of matrices that may be used for the formation ofdonor tissues or organs include collagen matrices, carbon fibers,polyvinyl alcohol sponges, acrylateamide sponges, fibrin-thrombin gels,hyaluronic acid-based polymers, and synthetic polymer matricescontaining polyanhydride, polyorthoester, polyglycolic acid, or acombination thereof (see, for example, U.S. Pat. Nos. 4,846,835;4,642,120; 5,786,217; and 5,041,138).

The RPSCs produced according to the invention may be used to producegenetically engineered or transgenic differentiated cells. Essentially,this will be effected by introducing a desired gene or genes, orremoving all or part of an endogenous gene or genes of RPSCs producedaccording to the invention, and allowing such cells to differentiateinto the desired cell type. A preferred method for achieving suchmodification is by homologous recombination because such technique canbe used to insert, delete or modify a gene or genes at a specific siteor sites in the stem-like cell genome.

This methodology can be used to replace defective genes, e.g., defectiveimmune system genes, cystic fibrosis genes, or to introduce genes whichresult in the expression of therapeutically beneficial proteins such asgrowth factors, lymphokines, cytokines, enzymes, etc. For example, thegene encoding brain derived growth factor maybe introduced into humanembryonic or stem-like cells, the cells differentiated into neural cellsand the cells transplanted into a Parkinson's patient to retard the lossof neural cells during such disease. Examples of mutations that may berescued using these methods include mutations in the cystic fibrosisgene; mutations associated with Dunningan's disease such as the R482W,R482Q, and R584H mutations in the lamin A gene; and mutations associatedwith the autosomal-dominant form of Emery Deyfuss muscular dystrophysuch as the R249Q, R453W, and Q6STOP mutations in the lamin A gene. Inthe Q6STOP mutation, the codon for Gln6 is mutated to a stop codon.

Previously, cell types transfected with BDNF varied from primary cellsto immortalized cell lines, either neural or non-neural (myoblast andfibroblast) derived cells. For example, astrocytes have been transfectedwith BDNF gene using retroviral vectors, and the cells grafted into arat model of Parkinson's disease (Yoshimoto et al., Brain Research,691:25-36, (1995)). This ex vivo therapy reduced Parkinson's-likesymptoms in the rats up to 45% 32 days after transfer. Also, thetyrosine hydroxylase gene has been placed into astrocytes with similarresults (Lundberg et al., Develop. Neurol., 139:39-53 (1996) andreferences cited therein).

However, such ex vivo systems have problems. In particular, retroviralvectors currently used are down-regulated in vivo and the transgene isonly transiently expressed (review by Mulligan. Science 260: 926-932(1993)). Also, such studies used primary cells, astrocytes, which havefinite life span and replicate slowly. Such properties adversely affectthe rate of transfection and impede selection of stably transfectedcells. Moreover, it is almost impossible to propagate a large populationof gene targeted primary cells to be used in homologous recombinationtechniques.

By contrast, the difficulties associated with retroviral systems shouldbe eliminated by the use of RPSCs of the present invention, which areES-like cells. Using known methods to introduced desired genes/mutationsinto ES cells, RPSCs may be genetically engineered, and the resultingengineered cells differentiated into desired cell types, e.g.,heniatopoietic cells, neural cells, pancreatic cells, cartilage cells,etc. Genes which may be introduced into the RPSCs include, for example,epidermal growth factor, basic fibroblast growth factor, glial derivedneurotrophic growth factor, insulin-like growth factor (I and II),neurotrophin3, neurotrophin-4/5, ciliary neurotrophic factor, AFT-1,cytokine genes (interleukins, interferons, colony stimulating factors,tumor necrosis factors (alpha and beta), etc.), genes encodingtherapeutic enzymes, collagen, human serum albumin, etc.

In addition, it is also possible to use one of the negative selectionsystems now known in the art for eliminating therapeutic cells from apatient if necessary. For example, donor cells transfected with thethymidine kinase (TK) gene will lead to the production of embryoniccells containing the TK gene. Differentiation of these cells will leadto the isolation of therapeutic cells of interest which also express theTK gene. Such cells may be selectively eliminated at any time from apatient upon gancyclovir administration. Such a negative selectionsystem is described in U.S. Pat. No. 5,698,446, and is hereinincorporated by reference.

Examples of diseases, disorders, or conditions that may be treated orprevented include neurological, endocrine, structural, skeletal,vascular, urinary, digestive, integumentary, blood, immune, auto-immune,inflammatory, endocrine, kidney, bladder, cardiovascular, cancer,circulatory, digestive, hematopoeitic, and muscular diseases, disorders,and conditions. In addition, reprogrammed cells may be used forreconstructive applications, such as for repairing or replacing tissuesor organs.

With respect to the therapeutic methods of the invention, it is notintended that the administration of RPSCs to a mammal be limited to aparticular mode of administration, dosage, or frequency of dosing; thepresent invention contemplates all modes of administration, includingintramuscular, intravenous, intraarticular, intralesional, subcutaneous,or any other route sufficient to provide a dose adequate to prevent ortreat a disease. The RPSCs may be administered to the mammal in a singledose or multiple doses. When multiple doses are administered, the dosesmay be separated from one another by, for example, one week, one month,one year, or ten years. One or more growth factors, hormones,interleukins, cytokines, or other cells may also be administered before,during, or after administration of the cells to further bias themtowards a particular cell type.

The RPSCs of the present invention may be used as an in vitro model ofdifferentiation, in particular for the study of genes which are involvedin the regulation of early development. Differentiated cell tissues andorgans using the RPSCs may be used in drug studies.

Furthermore, the RPSCs produced according to the invention maybeintroduced into animals, e.g., SCID mice, cows, pigs, e.g., under therenal capsule or intramuscularly and used to produce a teratoma therein.This teratoma can be used to derive different tissue types. Also, theinner cell mass produced by X-species nuclear transfer may be introducedtogether with a biodegradable, biocompatible polymer matrix thatprovides for the formation of 3-dimensional tissues. After tissueformation, the polymer degrades, ideally just leaving the donor tissue,e.g., cardiac, pancreatic, neural, lung, liver. In some instances, itmay be advantageous to include growth factors and proteins that promoteangiogenesis. Alternatively, the formation of tissues can be effectedtotally in vitro, with appropriate culture media and conditions, growthfactors, and biodegradable polymer matrices.

Applications of the Somatic Cell Reprogramming Methods and RPSCs inAnimals

The reprogramming methods disclosed herein may be used to generate RPSCsfor a variety of animal species. The RPSCs generated can be useful toproduce desired animals. Animals include, for example, avians andmammals as well as any animal that is an endangered species. Exemplarybirds include domesticated birds (e.g., quail, chickens, ducks, geese,turkeys, and guinea hens) as well as other birds such as birds of prey(e.g., hawks, falcons, ospreys, condors, etc.), endangered birds (e.g.,parrots, California condor, etc.), ostriches etc. Exemplary mammalsinclude murine, caprine, ovine, bovine, porcine, canine, feline andprimate. Of these, preferred members include domesticated animals,including, for examples, cattle, buffalo, pigs, horses, cows, rabbits,guinea pigs, sheep, and goats.

RPSCs generated by the reprogramming methods of the present inventionallows one, for the first time, to genetically engineer animals otherthan mouse and human. RPSCs are ES-like cells, and are thus amenable togenetic manipulation. To date, no ES cells are available, for animalsother than mouse and human. As a result, for these animals, it iscurrently practically impossible to create genetically modified animalshaving targeted mutations. The ES-cell like RPSCs can be manipulated tointroduce desired targeted genetic modifications. The resultingengineered RPSCs can then be used to generate a cloned animal with thedesired genetic modifications in its germ line, using methods describedfor ES cells in mouse. See Capecchi and Thomas, U.S. Pat. Nos.5,487,992, 5,627,059, 5,631,153, and 6,204,061. Genetic engineering inanimals has potentially great applications in a variety of animals,especially farm animals.

The somatic cell reprogramming methods of the present invention providesat least two methods for delivering optimized farm animals. In thefirst, somatic cell reprogramming can be used to capture the bestavailable phenotype for a farm animal stock. The current technologiesused to deliver optimized farm animals are based on selective breeding,and expansion from preferred breeding stocks. Animals that have beenselected on the basis of superior characteristics, including, forexample, meat content, egg production (in the case of poultry), feedconversion ratio, are used to breed large numbers of animals that are inturn used in the human food supply. This traditional process hasprofound inherent inefficiencies. The phenotype observed in anindividual animal is often only partially transmitted in the progeny ofthat animal. Therefore, traditional breeding schemes are inefficient incapturing the very best phenotype in all of the progeny animals. Incontrast, the reprogramming methods of the present invention provides acontrolled and efficient way to achieve the same goal, by generatingRPSCs from somatic cells of an animal with the desired characteristics.The RPSCs generated may be used immediately to generate cloned animalsderived from the RPSCs. Known methods for generating mice from ES cellscan be used for this procedure. Alternatively, the RPSCs generated maybe cryopreserved and thawed in response to a grower's needs.

In the second method, somatic cells from an animal with the desiredcharacteristics are reprogrammed to produce RPSCs. The RPSCs are furthergenetically engineered to introduce desired genetic modification(s),before being placed into a recipient embryo to produce desired progeny.

The reprogramming methods can also be used to rescue endangered species.Somatic cell reprogramming provides an efficient method to generateRPSCs from somatic cells of an endangered animal. The resulting RPSCscan be used immediately to expand the numbers of the endangered animal.Alternatively, the RPSCs can be cryopreserved to generate a RPSC stockfor the endangered species, as a safeguard measure against extinction ofthe endangered species.

Methods for Gene Identification

The present invention provides methods for identifying a gene thatactivates the expression of an endogenous pluripotency gene in somaticcells. The methods comprise: transfecting the somatic cells of thepresent invention with a cDNA library prepared from ES cells or oocytes,selecting for cells that express the first selectable marker, andassessing the expression of the first endogenous pluripotency gene inthe transfected cells that express the first selectable marker. Theexpression of the first endogenous pluripotency gene indicates that thecDNA encodes a gene that activates the expression of an endogenouspluripotency gene in somatic cells.

The methods are applicable for identifying a gene that activates theexpression of at least two endogenous pluripotency genes in somaticcells. The somatic cells used in the methods further comprise a secondendogenous pluripotency gene linked to a second selectable marker. Themethods are modified to select for transfected cells that express bothselectable markers, among which the expression of the first and thesecond endogenous pluripotency genes are assessed. The expression ofboth the first and the second endogenous pluripotency genes indicatesthat the cDNA encodes a gene that activates the expression of at leasttwo pluripotency genes in somatic cells.

The methods are further applicable for identifying a gene that activatesthe expression of at least three endogenous pluripotency genes insomatic cells. The somatic cells used in the methods further comprise athird endogenous pluripotency gene linked to a third selectable marker.The methods are modified to select for transfected cells that expressall three selectable markers, among which the expression of all threeendogenous pluripotency genes are assessed. The expression of all threeendogenous pluripotency genes indicates that the cDNA encodes a genethat activates the expression of at least three pluripotency genes insomatic cells.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of mouse genetics, developmentalbiology, cell biology, cell culture, molecular biology, transgenicbiology, microbiology, recombinant DNA, and immunology, which are withinthe skill of the art. Such techniques are described in the literature.See, for example, Current Protocols in Cell Biology, ed. by Bonifacino,Dasso, Lippincott-Schwartz, Harford, and Yamada, John Wiley and Sons,Inc., New York, 1999; Manipulating the Mouse Embryos, A LaboratoryManual, 3^(rd) Ed., by Hogan et al., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 2003; Gene Targeting: A PracticalApproach, IRL Press at Oxford University Press, Oxford, 1993; and GeneTargeting Protocols, Human Press, Totowa, N.J., 2000. All patents,patent applications and references cited herein are incorporated intheir entirety by reference.

EXEMPLIFICATION

The invention now being generally described, it will be more readilyunderstood by reference to the following example, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

Example Oct4-Induced Fibroblasts are More Susceptible to Reprogrammingthan Unduced Fibroblasts as Demonstrated by Nuclear Transfer ExperimentA. Generation of Transgenic Mouse Carrying an Inducible Oct4 Transgene

An inducible Oct4 allele is constructed as the following: first, twointegration vectors are constructed. The first integration vector,inducible Oct4 integration vector, contains an Oct4 gene driven by atetracycline-inducible promoter (Tet-Op). The Tet-Op-Oct4 cassette isflanked by a splice-acceptor double poly-A signal (SA-dpA) at its 5′ endand a SV40 polyA tail (SV40-pA) at its 3′ end. The second integrationvector, tetracycline activator integration vector, contains a mutantform of tetracycline activator, M2-rtTA, which is more responsive todoxycycline (Dox) induction than the wild type activator. (Urlinger S.et al., 2000)

The two integration vectors are introduced into V6.5 ES cells: theinducible Oct4 integration vector and the tetracycline activatorintegration vector are introduced into the Collagen locus and the Rosa26locus respectively via site-specific integration, as shown in FIG. 1.The resulting ES cells are used to make Oct4-inducible mice bytetraploid complementation.

B. Expression of the Inducible Oct4 Transgene

Fibroblasts derived from tail biopsies of the Oct4-inducible mice werecultured. A fraction of the cultured fibroblasts were induced withdoxycycline for 3 days (at 2 microgram/ml), and Oct4 expression wasdetected by Northern blot and Western blot analysis. As shown in FIG. 2,the Oct4 expression level in fibroblasts treated with doxycycline iscomparable to the Oct4 expression level in ES cells, and undetectable infibroblasts not treated with doxycycline. The expression resultsdemonstrate that the inducible Oct4 transgene is expressed as planned.

C. Nuclear Transfer Experiment

Nuclear transfer was performed on fibroblasts derived from tail biopsiesof mice that carry the inducible Oct4 transgene. Dox induction was for24 hours prior to nuclear transfer. Cloned embryos were then activatedand cultured to the blastocyst stage to derive ES cells as describedpreviously (Hochedlinger and Jaenisch, 2002). As shown in Table 1, onaverage, blastocyst formation and ES cell derivation (as measured as afraction of eggs with pronucleus formation) is more efficient from Oct4induced fibroblast than from uninduced fibroblasts. This resultdemonstrated that induced Oct4 expression in somatic cells such asfibroblasts make these cells more susceptible to reprogramming.

One skilled in the art readily appreciates that the present invention iswell adapted to carry out the objects and obtain the ends and advantagesmentioned, as well as those inherent therein. The methods, systems andkits are representative of preferred embodiments, are exemplary, and arenot intended as limitations on the scope of the invention. Modificationstherein and other uses will occur to those skilled in the art. Thesemodifications are encompassed within the spirit of the invention and aredefined by the scope of the claims. It will be readily apparent to aperson skilled in the art that varying substitutions and modificationsmay be made to the invention disclosed herein without departing from thescope and spirit of the invention.

TABLE 1 In vitro development of clones derived from Oct4-inducedfibroblasts eggs w/ Blastocysts ES lines Expt. Oct4 PN (% PN) (% PN) #1− 22  5 (23%) 0 (0%) {close oversize brace} 19% {close oversize brace}3% #2 − 35  5 (14%) 2 (6%) #3 + 37 10 (27%) 2 (5%) {close oversizebrace} 24% {close oversize brace} 7% #4 + 47 10 (21%) 4 (9%) PN . . .ProNucleus formation Nuclear transfer was performed on fibroblastsderived from tail biopsies of mice that carry the inducible Oct4transgene. Dox induction was for 24 hours prior to nuclear transfer.Cloned embryos were then activated and cultured to the blastocyst stageto derive ES cells as described previously (Hochedlinger and Jaenisch,Nature, 2002). These preliminary results show that on average blastocystformation and ES cell derivation is more efficient from Oct4 inducedthan from uninduced fibroblasts.

REFERENCES

-   Avilion, J., et al., Nat. Biotechnol. 20: 1240-45 (2003)-   Gossen M. et al., Transcriptional activation by tetracyclines in    mammalian cells, Science 268: 1766-1769 (1995).-   Hanna, L. A., et al., Genes Dev. 16: 2650-61 (2002).-   Hochedlinger and Jaenisch, Nature 415: 1035-1038 (2002).-   Hogan et al., Cold Spring Harbor Laboratory Press, Cold Spring    Harbor, N.Y., 2003-   Ihle, J. H., Cell 84: 331-334 (1996)-   Munsie M. J., et al., Curr. Biol. 10: 989 (2000).-   Shmblott, M. J., et al., Derivation of pluripotent stem cells from    cultured human primordial germ cells. Proc. Natl. Acad. Sci. USA 95:    13726-13731 (1998)-   Smith A. G., et al. Nature 336: 688-690 (1988)-   Tan, D. S., Foley, M. A., Shair, M. D. & Schreiber, S. L. J. Am.    Chem. Soc. 120, 8565-8566 (1998).-   Thomson, J. A., et al., Embryonic stem cell lines derived from human    blastocysts. Science, 282: 1145-1147 (1998).-   Urlinger S, Baron U, Thellmann M, Hasan M T, Bujard H, Hillen W.-   Proc. Natl. Acad. Sci. USA. 97(14):7963-8 (2000). Exploring the    sequence space for tetracycline-dependent transcriptional    activators: novel mutations yield expanded range and sensitivity.-   William R. L., et al., Nature 336: 684-687 (1988)-   Yamada, Y., Miyashita, T., Savagner, P., Horton, W., Brown, K. S.,    Abramczuk, J., Xie, H. X., Kohno, K., Bolander, M. and Bruggeman, L.    (1990). Regulation of the collagen II gene in vitro and in    transgenic mice. Ann. New York Acad. Sci. 580, 81-87-   Zambrowicz B. P. et al., Disruption of overlapping transcripts in    the ROSA bgeo 26 gene trap strain leads to widespread expression of    b-galatosidase in mouse embryos and hematopoietic cells. Proc. Natl.    Acad. Sci. USA 94: 3789-3794 (1997).

1.-16. (canceled)
 17. A primary somatic cell comprising a firstendogenous pluripotency gene operably linked to DNA encoding a firstselectable marker in such a manner that expression of the firstselectable marker substantially matches expression of the firstendogenous pluripotency gene, wherein the cell comprises an exogenouslyintroduced candidate agent of interest with respect to its potential toreprogram a somatic cell, and wherein the pluripotency gene is a genethat is expressed in a pluripotent embryonic stem cell, is required forits pluripotency, and is downregulated as the cells differentiates. 18.The primary somatic cell of clam 17, further comprising a secondendogenous pluripotency gene operably linked to DNA encoding a secondselectable marker in such a manner that expression of the secondselectable marker substantially matches expression of the secondendogenous pluripotency gene.
 19. The primary somatic cell of claim 18,further comprising a third endogenous pluripotency gene operably linkedto DNA encoding a third selectable marker in such a manner thatexpression of the third selectable marker substantially matchesexpression of the third endogenous pluripotency gene.
 20. The primarysomatic cell of claim 17, wherein the candidate agent is a small organiccompound.
 21. The primary somatic cell of claim 17, wherein thecandidate agent is a peptide.
 22. The primary somatic cell of claim 17,wherein the candidate agent is a nucleic acid.
 23. A primary somaticcell comprising a first endogenous pluripotency gene operably linked toDNA encoding a first selectable marker in such a manner that expressionof the first selectable marker substantially matches expression of thefirst endogenous pluripotency gene, wherein the pluripotency gene is agene that is expressed in a pluripotent embryonic stem cells and whoseexpression is required for its pluripotency, and is downregulated as thecells differentiates, and wherein the position of the DNA encoding theselectable marker is outside the open reading frame of the endogenouspluripotency gene.
 24. The primary somatic cell of claim 23, furthercomprising an exogenously introduced pluripotency gene, wherein thepluripotency gene is a gene that is expressed in a pluripotent embryonicstem cell, is required for its pluripotency and is downregulated as thecell differentiates.
 25. The primary somatic cell of claim 24, whereinthe exogenously introduced pluripotency gene is Oct4. Nanog, or Sox2.26. The primary somatic cell of claim 23, further comprising anexogenously introduced candidate agent of interest with respect to itspotential to reprogram a somatic cell.
 27. The primary somatic cell ofclaim 23, wherein the endogenous pluripotency gene encodes Oct4 orNanog.
 28. The primary somatic cell of claim 17, wherein the endogenouspluripotency gene encodes Oct4 or Nanog.
 29. The primary somatic cell ofclaim 17, wherein the candidate agent is a candidate pluripotencyprotein.
 30. The primary somatic cell of claim 29, wherein thepluripotency protein is Oct4, Nanog, or Sox2.
 31. The primary somaticcell of claim 22, wherein the nucleic acid comprises a candidatepluripotency gene, wherein the candidate gene is a gene that isexpressed in a pluripotent embryonic stem cell, is required for itspluripotency, and is downregulated as the cell differentiates.
 32. Theprimary somatic cell of claim 31, wherein the pluripotency gene encodesOct4, Nanog, or Sox2.
 33. The primary somatic cell of claim 20, whereinthe small or organic compound is a synthetic compound.
 34. The primarysomatic cell of claim 17, wherein the cell is a mammalian cell.
 35. Theprimary somatic cell of claim 23, wherein the cell is a mammalian cell.36. A primary somatic cell comprising, a first endogenous pluripotencygene operably linked to DNA encoding a first selectable marker in such amanner that expression of the first selectable marker substantiallymatches expression of the first endogenous pluripotency gene, whereinthe first endogenous pluripotency gene encodes Oct-4 or Nanog, andwherein the cell further comprises an exogenously introduced Oct4,Nanog, or Sox2 protein, or a nucleic acid encoding Oct4, Nanog, or Sox2.37. The primary somatic cell of claim 36, further comprising a candidateagent of interest with respect to its potential to reprogram a somaticcell.
 38. A composition comprising: (i) a primary somatic cellcomprising a first endogenous pluripotency gene operably linked to DNAencoding a first selectable marker in such a manner that expression ofthe first selectable marker substantially matches expression of thefirst endogenous pluripotency gene; and (ii) a candidate agent ofinterest with respect to its potential to reprogram a somatic cell,wherein the first endogenous pluripotency gene encodes Oct4 or Nanog.39. The composition of claim 38, wherein the cell further comprises anexogenously introduced Oct4, Nanog, or Sox2 protein, or a nucleic acidencoding Oct4, Nanog, or Sox2.
 40. The primary somatic cell of claim 38,wherein the cell is a mammalian cell.